PROJECTSUMMARY A central goal in HIV/AIDS vaccine research is theelicitation of broadlyneutralizingantibodies (bNAbs). Here, weproposetoleveragethreerecentdiscoveriesfromourgroupstogeneratenovelenvelope(Env)immunogens that target the V1V2 region of the trimer apex. By studying the evolution of the HIV-1 Env glycan shield, we discoveredthatunshieldedregions(?glycanholes?)intransmittedfounder(TF)Envsarenegativelyassociated with bNAb development, suggesting that strain-specific glycan holes delay or subvert bNAb development (1). GeneratingbNAbsensitivitysignaturestodesignnovelSignature-basedEpitopeTargeted(SET)vaccines,we foundthatV2-SETimmunogensinducedbroaderandmorepotenttier2heterologousNAbsinguineapigsthan wild-type Envs, indicating that inclusion of bNAb signatures significantly improved vaccine performance (2). Studying20novelSHIVsin~100rhesusmacaques(RMs)(3),wefoundthat~15%ofanimalsdevelopedvarying degreesofheterologousbreadthby6-24months,withthemostcommonbNAbspecificitytargetingtheV2apex (Table 1). However, bNAb induction in SHIV infection is still infrequent, thus providing a unique experimental setting to test iterative Env design improvements in a manner that is faster and less costly than human trials. Ourhypothesisisthatby(i)minimizingdistractingglycanholeepitopes,(ii)increasingEnvaffinityforV2apex bNAbprecursors,(iii)increasingrelevantepitopediversityinvaccineboosts,and(iv)incorporatingBcelllineage immunogendesigns, we will improveV2 bNAb germlineengagement andbNAb lineage maturation. Wehave selected the CRF.AG.T250 (T250) and CAP256SU (CAP256) Envs as baseline immunogens, because both have generated V2 apex bNAbs in SHIV infected RMs (Table 1). In Aim #1, we will optimize the Env glycan shieldandV2germlinetargetingpropertiesofT250andCAP256Envs,testtheirreplicationpotentialandtier2 antigenicityinSHIVvectors,anddown-selectthebestperformingsetforsubsequentinfectionofRMs.InAim #2,wewillcomparethebNAbinductioncapacityofSHIVsexpressingwildtype(WT),glycan-optimized(GLY- OPT),andglycanandgermline-optimized(GLY/UCA-OPT)versionsofthesameEnvinRMsanddeterminethe envelope-antibody(Env-Ab)coevolutionpathwaysinallanimalsthatdevelopneutralizationbreadth.InAim#3, we will rationally design new V2 apex directed immunogens using Env-Ab co-evolution data and the V2-SET strategy as a guide, and deliver them using nucleoside-modified mRNA containing lipid nanoparticles (mRNA/LNPs)thatexpress stabilized, membrane bound gp160s. We will prime RMs with the bestperforming EnvfromAim#2,andthencomparethebNAbinductioncapacityofthisEnvwiththatoftwoboostingregimens specifically designed to increase relevant epitope diversity. Immunized RMs will receive a low-dose repetitive rectalSHIVchallengetoassesstheirlevelofprotectionasrecentlydescribed(4)andtostudytheirbreakthrough infections.Bysimultaneouslyapplyingmultiplevaccineimprovementstrategies,weexpecttoimproveV2apex bNAbinductioninRMsandtranslatethesefindingsintomoreeffectivevaccinesforhumans.